Fluorescence detection

ABSTRACT

The present invention provides methods of detecting and/or characterizing the viral vector particle content of a medium. A medium is provided and contacted with an excitation energy such that, if a viral vector particle is in the medium, an electron associated with the intrinsically fluorogenic portion of the viral vector particle will be raised to an excited energy state. The excited electron is permitted to emit radiation having an emission wavelength which is detected. The viral vector particle content of the medium then can be evaluated by comparing the detected emission wavelength with a standard signal. For example, the number of viral vector particles in a medium can be quantified by comparing the detected wavelength and its corresponding intensity to a standard signal. Similar methods for evaluating the adenoviral vector particle content of a medium and the intrinsically fluorogenic adenoviral structural protein content of a medium are provided.

TECHNICAL FIELD OF THE INVENTION

[0001] This invention pertains to the detection and characterization ofviral vector particles, particularly through the use of fluorescence.

BACKGROUND OF THE INVENTION

[0002] Viral vectors are of significant importance in several aspects ofmolecular biology and medicine. Numerous types of viral vectors havebeen developed for use as gene delivery vehicles. Examples of such viralvectors include vectors based on adenovirus (Ad), adeno-associated virus(AAV), baculovirus, herpes simplex virus (HSV), and murine leukemiavirus (MLV). Compared to other methods of delivering genetic information(e.g., lipo some-associated delivery techniques or naked DNA vectors),viral vectors offer several advantages, including higher rates ofdelivery and better targeting of specific tissues and/or cells. With theincreased use of viral vectors for therapeutic, as well as diagnostic,applications there is an increasing need for better methods forquantification and characterization of viral vector particles.

[0003] Several techniques are known for the characterization and/orquantification of viral vector particles, including chromatographicmethods and mass spectrometry (see, e.g., International PatentApplication WO 99/54441, International Patent Application WO 00/40702,and U.S. Pat. No. 5,965,358). Presently, the quantification of viralvector particles is most commonly carried out by the use of ultraviolet(UV) radiation. For example, U.S. Pat. No. 5,837,520 disclosesmonitoring the absorbance of a chromatographic eluant of viral particlesat a selected UV wavelength and comparing the absorbance value to astandard curve which relates absorbance to the number of viral vectorparticles. Ultraviolet absorbance is limited in its sensitivity andrequires a large number of viral particles (typically about 5×10⁹particles/ml) for accurate detection (where the standard deviation inmeasurement is about 10% or less). Due to the large number of viralparticles required for accurate quantification, ultraviolet absorbanceis not useful in applications requiring small populations of viralparticles, such as viral vector-based gene therapies where highquantities of viral vector particles can be undesirable.

[0004] Fluorescence-based detection and quantification of viralparticles associated with fluorogenic dyes such as fluoresceinisothiocynate (FITC) is known in the art. For example, Hara et al.,Applied and Environmental Microbiology, 57(9), 2731-34 (1991), describesthe use of epifluorescent microscopy on DAPI(4′,6′-diamidino-2-phenylindole)-treated water samples to determinenumbers of bacteria, viruses, and DNA-associated particles. Morerecently, Hennes and Suttle, Limnol. Oceanogr., 10(6), 1050-55 (1995),describes similar research using the cyanine-based dye, Yo-Pro-1.

[0005] Immunofluorescence, which combines antibody-antigen binding andfluorophore-associated fluorescence detection (see, e.g., Tanaka et al.,J. Hepatology, 23, 742-45 (1995)), also has been used to detect and/orcharacterize viruses. For example, D'alessio et al., AppliedMicrobiology, 20(2), 233-39 (1970), discloses the use offluorescein-based immunofluorescence techniques to detect influenzaviruses, herpes simplex virus, and adenoviruses. More recently, Orito etal., Gut, 39, 876-880 (1996), described the use of a fluorescent enzymeimmunoassay (FEIA) to quantify hepatitis C virus core protein levels inpatients, and Wood et al., J. Medical Virol, 51, 198-201 (1997),describes the use of FITC-based immunofluorescence to identify and typeadenovirus isolates. Enzymatic techniques associated with fluorogenicdyes also are capable of detecting nucleic acids (see, e.g., U.S. Pat.No. 5,830,666).

[0006] The Green Fluorescent Protein (GFP), obtained from the jellyfishAequorea victoria (see, e.g., Prasher et al., Gene, 111, 229-33 (1992)),which is intrinsically fluorogenic, has been used to characterizeviruses by causing viruses to express GFP.

[0007] For example, International Patent Application WO 00/08182describes preparations of herpes virus expressing GFP fusion proteins todetect the progress of cell infection by the virus and to screen forneutralizing antibodies or inhibitors of infection. International PatentApplication WO 99/54348 discloses the use of vectors transfected withshort-lived GFP variants to assay activation or deactivation ofpromoters. International Patent Application WO 99/43843 teachestransfection with adenovirus vectors encoding GFP and tracking viralproduction by GFP-associated fluorescence.

[0008] Techniques for detecting or characterizing viral vector particlesbased on direct fluorescent dye-association with the viral particles,immunofluorescence, and GFP-associated viral fluorescence are limited inrequiring either a fluorogenic dye or GFP to be associated with theviral vector particles. Because the use of fluorogenic dyes can beexpensive, less sensitive than other techniques, and damaging tosamples, direct dye-association techniques are often unsuitable.Immunofluorescence, while more sensitive than direct dye-basedtechniques, requires specific epitopes and antibodies. GFP-basedtechniques require either chemical or genetic modification to associatethe viral vector particles with GFP.

[0009] Few fluorogenic methods have been used to study biologicalmaterials without the use of dyes or strong fluorogenic proteins such asGFP. U.S. Pat. No. 5,623,932 discloses the use of direct fluorogenicmethods to differentiate between normal and abnormal cervical tissues.The '932 patent discloses using laser-induced fluorescence (LIF) toidentify fluorogenic spectra associated with healthy tissue, relying onoxy-hemoglobin and NADH in the tissue as fluorophores, and further usingsuch spectra to identify “abnormal” tissues by comparing spectra. The'932 patent suggests that such abnormal tissue could be inflamed orinfected with human papilloma virus (HPV). However, the '932 patentfails to identify, characterize, or quantify HPV particles in suchtissues.

[0010] Accordingly, there remains a need for techniques which allow forimproved detection and characterization of viral vector particles. Thepresent invention provides methods for such detection andcharacterization through fluorescence detection of viral vectorparticles and viral vector proteins. These and other advantages of thepresent invention, as well as additional inventive features, will beapparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention provides a method of quantifying the numberof viral vector particles in a medium. A medium containing a viralvector particle having an intrinsically fluorogenic portion is provided.The medium is contacted with an excitation energy, such that an electronassociated with the intrinsically fluorogenic portion of the viralvector particle is raised to an excited energy state. The excitedelectron is permitted to emit radiation having one or more emissionwavelengths and corresponding emission intensities. An emissionwavelength, and the intensity of the emitted radiation at the emissionwavelength, are detected. The number of viral vector particles in themedium is quantified by evaluating the detected wavelength and intensityand comparing them to a provided standard signal.

[0012] The invention also provides a method of evaluating the viralvector particle content of a medium. A medium is provided and contactedwith an excitation radiation having an excitation wavelength such thatif a viral vector particle is in the medium an intrinsically fluorogenicportion of the viral vector particle will emit radiation having anemission wavelength at about 560-590 nm (e.g., about 575 nm). The viralvector particle content of the medium is evaluated by determiningwhether the medium emits radiation at about 560-590 nm.

[0013] The invention further provides a method of evaluating theadenoviral vector particle content of a medium. A medium is provided andcontacted with an excitation radiation having one or more excitationwavelengths suitable for exciting an electron associated with theintrinsically fluorogenic portion of an adenoviral vector (typically atabout 235 nm, about 284 nm, or both). If an adenoviral vector particleis in the medium, an intrinsically fluorogenic portion of the adenoviralvector particle will emit radiation having an emission wavelengthcharacteristic of a naturally-occurring (i.e., wild-type) adenoviralvector (typically at about 330 nm, about 574 nm, or both). Theadenoviral vector particle content of the medium is evaluated bydetermining whether the medium emits radiation having such an emissionwavelength (or wavelengths).

[0014] The invention also provides a method of evaluating theintrinsically fluorogenic adenoviral structural protein content of amedium. Similar to the other aspects of the invention, a medium isprovided and contacted with an excitation radiation having an excitationwavelength, such that if an intrinsically fluorogenic adenoviralstructural protein is in the medium it will emit radiation having anemission wavelength characteristic of an intrinsically fluorogenicwild-type adenoviral structural protein or a substantial homologthereof. The intrinsically fluorogenic adenoviral structural proteincontent of the medium is evaluated by determining whether radiationhaving an emission wavelength characteristic of an intrinsicallyfluorogenic wild-type adenoviral structural protein or substantiallyhomologous protein is emitted from the medium.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides methods of detecting and/orcharacterizing (e.g., quantifying) the viral vector particle content ofa medium. A medium in the context of the present invention is any mediumwhich is suitable for detection of radiation emitted from a wild-typeintrinsically fluorogenic portion (or portions) of a viral vector, orsubstantial homolog thereof, using the disclosed inventive methods. Themedium can include different types of intrinsically fluorogenic viralvector particles, other fluorogenic molecules and non-fluorogenicmolecules. The medium can take any suitable form. Typically, the mediumwill include or be in the form of a liquid, such as an aqueous solution.Such solutions can consist of numerous additional components, such asbuffers, stabilizers, preservatives, excipients, carriers, diluents, orother additives. The medium can comprise a pharmaceutically acceptable(e.g., a physiologically acceptable) carrier and can take the form of apharmaceutical composition. The medium can include one or more cells.For example, the medium can be a culture of cells, or a tissue, which iseither in a tissue culture or in an animal (e.g., an organ in a human).The medium can consist of a sample of a larger composition, such as apool or stock of viral vector particles (e.g., a library of viral genetransfer vector particles in a stock).

[0016] The medium can, and typically will, contain a viral vectorparticle having an intrinsically fluorogenic portion. The invention canbe practiced with any suitable type of viral vector particle. A viralvector particle is any molecule which is based upon, derived from, ororiginates from a virus, and which includes more than one type of viralmolecule (e.g., more than one type of viral protein or a viral proteinand a viral nucleic acid) or a substantial homolog thereof (as definedfurther herein). A viral molecule is any molecule which makes up aportion of a wild-type virus or a substantial homolog thereof.

[0017] The viral vector particle can be an unmodified naturallyoccurring (i.e., “wild-type”) virus particle, or modified viral vectorparticle, such as a viral gene transfer vector and/or a synthetic viralvector particle. Desirably, the viral vector particle contains, or isassociated with, a nucleotide genome. Preferably, though notnecessarily, the viral vector particle is derived from, is based on,comprises, or consists of, a virus which normally infects animals, suchas mammals and, especially, humans. Preferred types of viral vectorparticles include baculovirus vectors, herpes vectors, retroviralvectors, adeno-associated viral vectors, and adenoviral vectors.Adenoviral vector particles are particularly preferred.

[0018] The inventive method can be practiced with a medium containingany suitable number of viral vector particles. A suitable number ofviral vector particles is any number which can be detected and/orcharacterized (e.g., quantified) by the methods of the presentinvention. The inventive methods can be practiced with a homogenous orheterogeneous (i.e., mixed) population of viral vector particles (e.g.,wild-type herpes virus and adenovirus particles, or different modifiedparticles such as replication defective adenoviral vector particles andcomplementing (i.e., helper) adenovirus particles). When a number ofidentical or similar viral vector particles are present in a suitablemedium, the particle-containing medium can be referred to as a stock ofthe viral vector.

[0019] The viral vector particle can have any suitable size and weight.In contrast to UV spectrophotometry-based techniques, viral vectorparticles with relatively larger molecular weights and sizes can bedetected, characterized, and/or quantified directly using the methodsdescribed herein, without performing calculations or taking additionalsteps to account for scattered light problems associated with UV-baseddetection which may result in erroneous detection readings. For example,viral vector particles having molecular weights of about 1×10⁸ Daltonsor more, about 1.5×10⁸ Daltons or more, and even about 1.7×10⁸ Daltonsor more (e.g., about 2×10⁸ Daltons or more) can be detected and/orcharacterized (e.g., quantified) directly (i.e., without takingadditional steps or performing calculations to account for lightscattering). Further in contrast to UV spectrophotometry-basedtechniques, viral vector particles with large particle sizes can bedirectly detected and/or characterized (e.g., quantified). For example,viral vector particles of at least about 40 nm in diameter, at leastabout 80 nm in diameter, at least about 120 nm in diameter, or larger,can be directly detected and/or characterized.

[0020] The viral vector particle includes an intrinsically fluorogenicportion. The intrinsically fluorogenic portion in the context of thepresent invention is any portion of a viral vector particle whichincludes, or consists of, a naturally-occurring (wild-type) viralmolecule (e.g., an intrinsically fluorogenic viral protein), or asubstantially homologous (preferably substantially identical) molecule,which is intrinsically fluorogenic. A molecule is “intrinsicallyfluorogenic” if it emits one or more emission wavelengths when contactedwith a suitable excitation energy in the absence of fluorescent dyesand/or conjugated fluorophores.

[0021] Typically, and preferably, the intrinsically fluorogenic portionincludes, or consists of, a wild-type viral molecule. In such aspects,the molecule can be any suitable type of molecule. Examples of suitablemolecules include viral proteins, including post-translationallymodified viral proteins (e.g., viral glycoproteins).

[0022] The intrinsically fluorogenic portion can include, or consist of,an intrinsically fluorogenic molecule that is at least substantiallyhomologous, preferably substantially identical, to an intrinsicallyfluorogenic wild-type viral molecule (e.g., a non-wild-type homolog of awild-type viral fluorogenic protein). As used herein, a substantiallyhomologous molecule is any molecule having at least about 70% amino acidsequence homology to another molecule (e.g., a wild-type viral protein),at least about 70% structural similarity to another molecule (e.g., awild-type viral protein), or both.

[0023] The intrinsically fluorogenic portion has at least about 70%(e.g., at least about 80%, at least about 85%, or at least about 90%)amino acid sequence homology to an intrinsically fluorogenic wild-typeviral molecule if at least about 70% of the amino acid residues in thesubstantially homologous molecule's amino acid sequence are identicalto, or differ by only conservative amino acid residue substitutionsfrom, the amino acid residues in the amino acid sequence of itswild-type counterpart when the sequences are compared in a manner whichmaximizes homology and/or identity. Conservative amino acid residuesubstitutions involve exchanging a member within one class of amino acidresidues for a residue that belongs to the same class. Homologousproteins obtained by conservative substitutions are expected tosubstantially retain the biological properties and function of thewild-type protein. The classes of amino acids and the members of thoseclasses are presented in Table 1. TABLE 1 Amino Acid Residue ClassesAmino Acid Class Amino Acid Residues Acidic Residues ASP and GLU BasicResidues LYS, ARG, and HIS Hydrophilic Uncharged Residues SER, THR, ASN,and GLN Aliphatic Uncharged Residues GLY, ALA, VAL, LEU, and ILENon-polar Uncharged Residues CYS, MET, and PRO Aromatic Residues PHE,TYR, and TRP

[0024] A substantially homologous molecule can include any suitablenumber of non-conservative amino acid residue substitutions. Preferably,aromatic residues (which are fluorogenic) remain conserved with respectto, and more preferably remain identical to, the aromatic residuesoccurring in the corresponding wild-type viral molecule.

[0025] One of ordinary skill will recognize that residue position ineither substantially homologous or substantially identical molecules mayvary from their wild-type counterpart molecule due to deletions oradditions of residues. Homology and/or identity in view of suchsubstitutions and deletions can be determined using commerciallyavailable sequence analysis/alignment software and/or other knowntechniques. Protean, sold by DNASTAR (Madison, Wis.), is an example ofsuitable commercially-available sequence analysis software.

[0026] Alternatively, or in addition, the intrinsically fluorogenicportion has at least about 70% (e.g., at least about 80%, at least about85%, or at least about 90%) structural similarity to an intrinsicallyfluorogenic wild-type viral molecule. Thus, the intrinsicallyfluorogenic portion can have a significantly different amino acidsequence from wild-type viral proteins if there exists such structuralsimilarity. For example, synthetic peptides and/or recombinantlyproduced peptides which have a structure that is substantially similarto the structure of wild-type adenovirus fiber protein are contemplated.Examples of such proteins include modified fiber proteins that containknob (or “head”) region or domain modifications as described in, e.g.,U.S. Pat. No. 5,846,782, and double-abated adenoviruses which containmodified penton proteins.

[0027] The percentage of structural similarity can be based on completeoverlap between the molecules, on a domain-by-domain basis, or,preferably, by both methods. Structural similarity between the moleculescan be determined by any suitable method. For example, the secondarystructure of two proteins can be determined and compared, e.g., by meansof performing surface probability comparisons using the amino acidsequences of both molecules. Alternatively, and preferably, the threedimensional structures for the two proteins are determined and compared(e.g., by overlapping the three dimensional structures of the proteinsusing three dimensional imaging software).

[0028] The intrinsically fluorogenic portion can be substantiallyidentical to an intrinsically fluorogenic wild-type viral molecule suchthat it has at least about 70% (preferably at least about 80%, and morepreferably at least about 90%) amino acid sequence identity with awild-type viral protein. Preferably, although not necessarily, theintrinsically fluorogenic portion also will have at least 70% structuralsimilarity to its wild-type viral counterpart.

[0029] The intrinsically fluorogenic portion desirably has at leastsimilar fluorogenic properties to its wild-type viral counterpart. Inother words, the intrinsically fluorogenic portion preferably emitsradiation having at least one emission wavelength in common with itswild-type viral counterpart.

[0030] The viral vector particle can include, and preferably doesinclude, more than one intrinsically fluorogenic portion, and eachintrinsically fluorogenic portion preferably includes more than oneintrinsically fluorogenic molecule (e.g., 3, 5, 10, 15, or moreintrinsically fluorogenic wild-type viral proteins). Additionally oralternatively, the viral vector particle can include non-natural and/ornon-viral fluorogenic portions in addition to the intrinsicallyfluorogenic portion (e.g., the viral vector particle can include GFP).

[0031] The intrinsically fluorogenic portion can make up any suitableportion of the viral vector particle, including the entire particle.Desirably, the molecules which make up the intrinsically fluorogenicportion (or portions) make up at least about 10%, preferably at leastabout 20%, more preferably at least about 50%, and even more preferablyat least about 70% of the molecules which form the viral vector (eitherby weight, molecule type, or both).

[0032] The viral vector particle-containing medium is contacted with anexcitation energy. The excitation energy can be any form of energy whichis capable of exciting an electron associated with an intrinsicallyfluorogenic portion of the viral vector particles to an excited energystate. While numerous forms of energy are suitable, the excitationenergy preferably is in the form of an excitation radiation.

[0033] Excitation radiation can be any suitable type of radiation.Typically, the excitation radiation will be in the form ofelectromagnetic radiation having one or more discrete wavelengths. Forexample, the excitation radiation can be in the form of visible lightradiation, such as is emitted from a suitable lamp. Alternatively, theexcitation radiation can be in the form of ultraviolet radiation (UV) orinfrared (IR) radiation. The excitation radiation can be generated byany suitable technique or device. Most often, the excitation radiationis in the form of one or more photons of energy supplied by a suitableradiation source, such as an incandescent lamp, argon/mercury lamp,xenon lamp, halogen lamp, or a laser (e.g., through a laser-inducedflash).

[0034] Due to the speed with which excitation occurs using a xenon lampor a laser, the excitation energy is preferably provided by one of thesetwo sources. Because of its high sensitivity, laser-induced fluorescence(LIF) is particularly preferred. In LIF, the medium typically isirradiated at one wavelength, usually in the UV spectral region, and theemission (fluorescent signal) is measured at a longer wavelength,usually at a higher UV wavelength or the violet-yellow/green region ofthe visible spectrum. The excitation source for molecular LIF typicallyis a tunable dye laser in the UV spectral region. In addition to UVradiation, LIF can utilize visible and/or near-IR excitation radiation,particularly with recently developed frequency doubling methods. Coolingof the medium (and thus the viral vector particle), for example bymolecular beams, free-jet expansions, and cryogenic glass or crystallinematrices, in LIF-based techniques, can remove spectral congestion andreduce the Doppler width of the transitions, thereby allowing forimproved detection. The laser used for LIF can be any suitable laser,including, for example, an argon-ion laser or a helium/neon laser.Preferred laser fluorometers are ZetaLIF fluorometers, available fromPicometrics (Ramonville, Saint Agne, France). Typically, in using LIFtechniques, the medium will be, or comprise, a sample (i.e., a portion)of a larger composition due to the tendency of LIF techniques to damagebiological samples, including viable viral vector particles. Due to thehigher sensitivity associated with LIF-based techniques, such samplescan be relatively small and consist of very few viral vector particles(e.g., samples containing about 1×10⁶ viral vector particles or less aresuitable), and the results of applying the method can be extrapolated toquantify or otherwise characterize or evaluate the viral vectorparticles in a significantly larger composition.

[0035] An electron associated with an intrinsically fluorogenic portionof the viral vector particle is raised to an excited energy state by thecontact of the excitation energy with the medium. The process of raisingthe electron to an excited energy state is known as excitation, and theelectron in such a state is referred to as an excited electron.Excitation in the context of the present inventive method can occur inany suitable manner. For example, excitation can occur through directcontact of the viral vector particle with the excitation energy, or,alternatively, through absorption of the excitation energy and transferthereof through the medium to the viral vector particle. Any suitablenumber of electrons can be raised to the excited state by the excitationenergy. Desirably, more than one electron is excited. Thus, each viralvector particle desirably includes an intrinsically fluorogenic portionassociated with more than one excited electron.

[0036] The excited energy state can be any suitable energy state whichis higher than the energy state which the electron occupied immediatelyprior to contact with the excitation energy. A suitable energy state isany energy state which causes the excited electron, if permitted, toemit radiation. Thus, the excited electron can be raised (i.e.,“boosted”) to the next highest energy state it can occupy (a firstexcited state (e.g., an S₁ state)), or to a higher energy state whichthe electron can occupy (a second or higher excited state (e.g., an S₂state)). An electron's energy state can be characterized based on thevibrational energy, the rotational energy, or both, associated with theenergy state. Several vibrational and rotational energy levels can existwithin an excited state.

[0037] A radiation wavelength associated with exciting an electron is anexcitation wavelength. The excitation wavelength associated with aparticular viral vector particle is dependent upon the fluorogenicproperties of the particular viral vector. The radiation wavelengthassociated with exciting the largest number of viral vector particles isthe optimum excitation wavelength. The optimum excitation wavelength canbe determined by determining the excitation wavelength associated withthe apex of the highest “peak” on a graph of the viral vector particle'sexcitation spectrum (i.e., a two-dimensional plot of either excitationenergies or wavelengths versus the intensity of the resulting emittedradiation).

[0038] The viral vector particle can have any suitable number ofassociated excitation wavelengths. Preferably, the viral vector particlehas more than one associated excitation wavelength. In such situations,quantification of the number of viral vector particles typically ispracticed using the optimum excitation wavelength, which provides thegreatest sensitivity and most accurate detection of viral vectorparticles. In some situations, however, using other excitationwavelengths is preferred. For example, an excitation energy associatedwith the viral vector particles, which does not excite other (i.e.,non-viral vector) fluorogenic components of the medium, can be used toprovide greater selectivity for the viral vector particles, even if theexcitation radiation is not the optimum excitation wavelength.

[0039] Excitation of other fluorogenic components of the medium also canbe avoided by the use of wavelength selectors, which are known in theart. A wavelength selector screens radiation, thereby permitting onlycertain wavelengths, or bands (i.e., ranges) of wavelengths, to contactthe medium. Any suitable type of wavelength selector can be used.Typical wavelength selectors include monochromators, bandpass filters(such as long pass and short pass filters), and cutoff filters. Amonochromator or a bandpass filter permits a range of wavelengths of anexcitation radiation to pass through and contact the medium whileblocking radiation at the other excitation wavelengths. A monochromatorincreases the intensity of the resulting fluorescent emissions byselecting for a range of excitation wavelengths. A cutoff filter blocksstray excitation radiation below a predefined cutoff point.Monochromators, bandpass filters, and other components can be includedwithin a fluorescence detection system. For example, the system caninclude one or more gratings which are designed to optimize excitationand/or emission wavelengths, alone or in combination with one or moremirrors for directing excitation radiation to the medium or a portionthereof.

[0040] After excitation, the excited electron, if permitted to, willemit radiation having one or more emission wavelengths. This phenomenonof emitting radiation by an excited electron concomitant with theexcited electron's return to a ground or relaxed state is known asfluorescence. The excited electron's emission of radiation (also knownas a fluorescent emission) permits the excited electron to enter anenergy state lower than the excited energy state (sometimes referred toas the ground or relaxed state), which typically is substantially equalto the energy state the electron was in prior to contact with theexcitation energy.

[0041] Fluorescent emissions are marked by brief excitation emissionperiods. Fluorescent emissions usually begin almost instantaneously uponabsorption of radiation at a suitable excitation wavelength. Afterexcitation, fluorescent emissions can occur for any suitable period.Preferably, fluorescent emissions occur for about 5×10⁻³ seconds orless. Typically, fluorescent emissions will occur within a period ofabout 1×10⁻⁵−1×10⁻⁹ seconds.

[0042] The emitted radiation can have any suitable number of emissionwavelengths. The magnitude of the emission wavelengths is dependent uponthe excitation wavelength and fluorogenic characteristics of the viralvector particle, particularly the energy levels available to the viralvector particle-associated excited electrons. Similar to excitationwavelengths, emission wavelengths can form an emission spectrum, whichcan be graphically represented as a plot of emission wavelengths versusfluorescence intensity.

[0043] Because of energy dissipation during absorption, the emissionwavelength or wavelengths typically are longer than the excitationwavelength or wavelengths. The difference in energy or wavelengthrepresented by the difference between the excitation wavelength and theemission wavelength (hν_(EX)-hν_(EM)) is known as the Stokes shift. Thisdifference in length of the excitation and emission wavelengths,represented by the Stokes shift, permits isolation of either excitationor emission radiation. Accordingly, viral vector particles associatedwith large Stokes shifts are preferred.

[0044] Fluorescent emissions, in the context of the present invention,can have any suitable characteristics. Preferably, the fluorescentemissions are distinguishable from phosphorescent emissions orluminescent emissions. Thus, for example, the production of fluorescentemissions compared to the use of UV spectrophotometry is relativelytemperature independent, except with regard to an increased probabilityof quenching associated with higher temperatures in some mediums.

[0045] The fluorescence process usually is cyclical. Thus, unless theintrinsic fluorogenic capacity of a viral vector particle isirreversibly destroyed in the initial excited state (for example by thephenomenon of photobleaching), a viral vector particle can be repeatedlyexcited and detected. Thus, the excitation of an electron associatedwith a viral vector particle can be repeated as desired depending uponthe ability of the viral vector particle to undergo repeatedexcitation/emission cycles.

[0046] Often, not all of the electrons initially excited return to thelower energy state by fluorescence. Other processes such as collisionalquenching, fluorescence energy transfer, and intersystem crossing alsocan depopulate the population of excited electrons. The ratio of thenumber of fluorescence emissions to the number of photons absorbed by aviral vector particle is the fluorescence quantum yield. Thus, thequantum yield measures the relative extent to which processes whichdepopulate the population of excited electrons occur.

[0047] In order to maximize the quantum yield, the methods of thepresent invention preferably are practiced while avoiding photobleaching(i.e., photodestruction) and quenching of the fluorogenic properties ofthe viral vector particle. Any suitable technique for avoidingphotobleaching and quenching can be utilized. Examples of suitabletechniques include avoiding high intensity excitation radiation,maximizing detection sensitivity (e.g., by using low-light detectiondevices such as CCD cameras, as well as high-numerical apertureobjectives, and the widest emission bandpass filters compatible withsatisfactory signal isolation), using antifade agents, and avoidingagents associated with collisional quenching, such as O₂ and heavy atomssuch as iodide. When the medium is in solution (e.g., a portion of acomposition subjected to a chromatography resin), degassing thecomposition to remove such agents, particularly oxygen, and therebyavoid collisional quenching, is particularly preferred. In livecell-containing mediums, vitamin C (ascorbic acid) often can be used toreduce photobleaching. Collisional and self-quenching also can often bereduced, if necessary, by reducing the concentration of the viral vectorparticles and/or other components in the medium. Quenching isconformation dependent. Thus, modifying the conformation of the viralvector particles also can affect the probability of quenching.Environmental factors, such as medium polarity, proximity andconcentrations of quenching species, and pH, also should be monitoredfor photobleaching effects.

[0048] The emitted radiation can be characterized on the basis of itsintensity (also sometimes referred to in the art as brightness). Thetotal intensity of viral vector particle emitted radiation in aparticular medium is a function of the intensity and wavelength of theexcitation radiation, the amount of viral vector particles present inthe medium, and the fluorogenic properties of the viral vectorparticles.

[0049] Two fluorogenic properties of the viral vector particles whichaffect intensity are the extinction coefficient and quantum efficiency.The extinction coefficient is the amount of radiation of a givenwavelength that is absorbed by the viral vector particle upon contactwith the excitation radiation. The quantum efficiency of the viralvector particle is its capacity to convert such absorbed radiation toemitted fluorescent radiation. The molar extinction coefficient of aviral vector particle is defined as the optical density of a one-molarsolution of viral vector particles through a one-cm radiation path.Emission intensity is proportional to both the quantum efficiency andextinction coefficient.

[0050] The emitted radiation, particularly the wavelength and intensityof the emitted radiation corresponding to at least one emissionwavelength, is detected. If the viral vector particle emits radiation atmultiple emission wavelengths, it is typically preferred that the methodincludes determining the intensity of emitted radiation at thosewavelengths as well. Any suitable number of emission wavelengths, andintensities corresponding to any number of emission wavelengths, can bedetected.

[0051] The emitted radiation can be detected using any suitabletechnique. Preferably, a fluorescence detector is used to detect theemitted radiation. Any suitable fluorescence detector can be used, andnumerous types are known and commercially available. Generally, afluorescence detector registers emission radiation, including emissionwavelengths, intensities, or both, and produces a recordable output,usually as an electrical signal or a photographic image. To aiddetection, the fluorescence typically interacts with an emissionwavelength selector (such as a monochromator or an interference filter)and then is detected by a radiation detector, such as a photodiode or aphotomultiplier tube (PMT). Other radiation detectors, such as a CCDcamera, also can be used.

[0052] Suitable fluorescence detectors include fluorometers (sometimesreferred to in the art as fluorimeters), spectrofluorometers, andmicroplate readers, which measure the average fluorescent properties ofthe medium; fluorescence microscopes, which resolve fluorescence as afunction of spatial coordinates in two or three dimensions; fluorescencescanners, which resolve fluorescence as a function of spatialcoordinates in two dimensions for macroscopic objects such aselectrophoresis gels, blots, and chromatograms; and flow cytometers,which measure fluorescence per particle in a flowing stream, allowingsubpopulations of viral vector particles in the medium to be identified,evaluated, and quantified. Each type of instrument produces differentmeasurement artifacts and makes different demands on the viral vectorparticles. For example, although photobleaching is often a significantproblem in fluorescence microscopy, it is not a major impediment in flowcytometry because the dwell time (how long the excitation beam continuesto illuminate the medium) of the individual viral vector particles cellsin the excitation beam in flow cytometry is short. PMTs can be useful inlow intensity applications such as fluorescence spectroscopy and areoften integrated into such devices; however, other radiation detectorsalso are suitable.

[0053] Preferably, the fluorescence detector provides continuous rangesof excitation and emission wavelengths, in contrast to laser scanningmicroscopes and flow cytometers, which presently typically requireexcitation at a single fixed wavelength. Fluorometers and/orspectrofluorometers, which provide such qualities, are preferredfluorescence detectors. Generally, a fluorometer is a fluorescencedetector which includes an excitation source, a sample cell for testingthe medium (which typically is a portion of a larger composition), and aradiation detector, such as a PMT.

[0054] As indicated above, scanning fluorescence techniques are usefulin many aspects of the invention. Examples of such techniques includemoving a laser over the medium or, alternatively, using a CCD camera tocollect the entire image at once. CCD camera techniques are faster andpotentially more sensitive than scanning, but often provide lowerresolution than PMT-based scanning techniques. Other detectionconfigurations have been developed using multiple lasers, rotatingmirrors, and mounts that fix the laser and detectors in a constantposition, each of which provides different and particular advantages.For example, certain configurations can permit the determination of theshape and/or weight of the viral vector particle (e.g., systems whichuse a fluorometer similarly to a light scatter detector).

[0055] Detecting fluorescent emissions sometimes can be compromised bybackground signals, which may originate from other medium constituents(sometimes referred to in the art as autofluorescence). Autofluorescencedesirably is minimized. Autofluorescence can be minimized by using asuitable wavelength selector, such as a filter that reduces thetransmission of background signals (e.g., a bandpass filter).Alternatively, in three-dimensional imaging systems, confocal opticsimprove resolution in the third dimension. Such systems irradiatesequentially each point in three-dimensional space. Collection opticscollect the signal from the irradiated point and reject any informationthat is out of focus. If the viral vector particles are associated withmultiple excitation wavelengths, the use of longer wavelengths also canassist in avoiding background fluorescence. Another way to improve thesignal is to increase the viral vector particle concentration; however,in most instances care should be taken to avoid quenching caused at veryhigh viral vector particle density.

[0056] There are numerous other ways to improve signal detection andevaluation. For example, the excitation radiation can be eliminated fromthe collection pathway by several methods, including orienting the pathof the excitation radiation so that the excitation radiation avoidscontacting the detection pathway and inserting bandpass filters into thedetection pathway to reject the excitation wavelength. Fluorescentsignal strength also can be improved by increasing the dwell time orrepetitively scanning the sample and mathematically processing thesignals to reduce random noise.

[0057] A standard signal can be provided, and the number of viral vectorparticles in the medium can be quantified, by comparing the intensity ofthe detected fluorescent emissions emanating from the viral vectorparticle or particles with the standard signal. The intensity of theradiation emanating from the viral vector particle or particles istypically proportional to the number of viral vector particles emittingradiation, thereby permitting relative quantification of the viralvector particles by comparison to the standard signal. In practice, aradiation detector (such as a PMT) which transmits a currentproportional to the intensity of the radiation detected by it can beused to determine the intensity.

[0058] Quantification can be performed under any suitable conditions.Typically and preferably, wavelength and intensity of the excitationradiation are held constant (for example, using a controlled laser lightsource) to ensure proportionality between intensity and the number ofviral vector particles. Dwell time also can affect the intensity of theemitted radiation and also should be kept constant when determiningintensity for quantification purposes.

[0059] The standard signal can be any suitable signal which permitsquantification of the number of viral vector particles in the medium.There are numerous techniques available for obtaining a suitablestandard signal. For example, a standard medium having a known viralvector particle content can be used to produce a standard signal (e.g.,a standard emission spectrum), which can be compared to the emissionspectrum of the medium.

[0060] The inventive method can quantify any suitable number of viralvector particles in any suitable concentration. Desirably, theproportionality of the number of viral vectors to emitted radiationintensity is maintained throughout a wide range of viral vector particleconcentrations, though this is sometimes not possible at particularlyhigh concentrations. One skilled in the art can determine the suitablequantifiable range of particle concentrations and particle numbers byroutine experimentation. For example, the minimum number of viral vectorparticles for use can be determined by adding viral vector particles toa medium containing no viral vector particles in a stepwise manner, andtesting for fluorescence detection after each addition. The maximumconcentration and/or particle number can be determined by continuing thesteps of stepwise addition of viral vector particles and fluorescencedetection until the substantially linear relationship between viralvector particle number and emitted radiation intensity is no longerobserved. The fluorescence detector, the type of excitation radiation,and type of viral vector particle, may impact on the range of viralvector particle numbers which can be quantified by the inventive method.

[0061] The inventive method can be practiced using mediums containingsignificantly smaller viral vector particle populations compared tothose which can be detected by UV spectrometry. For example, mediumswith viral vector concentrations of about 1×10¹⁰ particles/ml or less,about 1×10⁹ particles/ml or less, about 1×10⁷ particles/ml or less,about 5×10⁶ particles/ml or less, about 1×10⁵ particles/ml or less, oreven lower concentrations, can be quantified using the inventive method.The amount of viral vector particles required for quantification dependsupon the source of the excitation energy. For example, using a xenonlamp to generate the excitation energy permits quantification of about1×10⁷ particles/ml or less (e.g., about 5×10⁶ particles/ml or less),whereas using LIF to generate the excitation energy permitsquantification of about 1×10⁵ particles/ml or less (e.g., about 5×10⁶particles/ml or less).

[0062] While quantification can be performed with any suitable level ofaccuracy, the present invention offers methods where quantification atsuch low concentrations is possible with relatively (e.g., compared toUV spectrophotometry) high levels of accuracy. For example, the range oferror (or coefficient of variation) in the detected quantity of viralvector particles using the techniques described herein is typicallyabout 15% or less, preferably about 10% or less, even more preferablyabout 5% or less, and optimally about 3% or less.

[0063] In some instances, fluorescence detection by UVspectrometry-based techniques can be desirable. For example, when themethods described herein are used to detect viral vector particles byfluorescence detection and the medium contains a very large populationof viral vector particles (e.g., about 1-2×10¹⁰ particles or more), UVspectrometry-based quantification of viral vector particles may bedesirable.

[0064] As indicated above, the inventive method can be practiced usingmediums consisting of a crude cell lysate of viral vector infected cellsor with purified lysates. Purification can significantly improve theaccuracy of quantification and remove improperly processed (i.e., emptyor defective) or otherwise damaged viral vector particles. Any suitabletechnique for purification can be used. Examples of suitablepurification techniques include chromatographic purification (e.g.,anion exchange chromatography purification), filtration purification(e.g., tangential flow ultrafiltration), and density gradientpurification (e.g., cesium chloride (CsCl) density gradientpurification). Such techniques can be combined or repeated as desired.

[0065] Purification by chromatography is preferred. Any suitable type ofchromatographic purification can be used. Preferably, chromatographypurification is performed using the anion exchange chromatographymethods described in International Patent Application WO 99/54441.Desirably, the medium is provided by contacting chromatography resinwith a composition comprising a viral vector particle, and eluting atleast a portion of the composition containing the viral vector particlefrom the chromatography resin, such that the time of elution of theviral vector particle from the chromatography resin is determinable. Thetime of elution of the viral vector particle provides another tool forevaluating the viral vector particle content of the medium.Particularly, by separating a viral vector particle containingcomposition on the basis of elution from a chromatography resin andapplying the inventive method to one or more of the portions of theseparated composition (i.e., treating each portion as a separate mediumfor purposes of the inventive method), one can distinguish between theviral vector particle and other fluorogenic components of the mediumexhibiting similar emission wavelengths on the basis of their respectiveelution times. Moreover, when the expected time of elution of the viralvector particle is known, such techniques provide a way to ensure that adetected fluorescent emission is associated with the viral vectorparticle, by comparing the observed time of elution with a standard(e.g., expected) time of elution. In addition, graphing elution timeagainst fluorescence intensity provides an elution spectrum. Suchelution spectrums can be used for relative quantification purposes.Fluorescence detection can be performed directly on the portion(s) ofthe composition suspected of containing the viral vector particle assuch portion(s) elute from the chromatography resin.

[0066] Purified mediums, when used, can be purified to any suitablelevel. Preferably, a purified medium is at least as pure as a lysate ofviral vector infected cells subjected to 1×CsCl density gradientpurification. More preferably, the medium is at least as pure as alysate subjected to 2×(i.e., twice repeated) CsCl density gradientpurification, and even more preferably is substantially as pure as a3×CsCl density gradient purified lysate. Examples of techniques forachieving high levels of purification are described, for example, inInternational Patent Application WO 99/54441.

[0067] The present invention also provides a method of quantifying thenumber of damaged viral vector particles, such as the number ofdefective viral vector particles, empty viral vector particles, or both,in the medium, by fluorescence detection. Defective viral vectorparticles are viral vector particles which are incompletely processed(i.e., contain one or more incompletely processed components such thatthey are not as intact as a fully processed viral vector particle).Empty viral vector particles are particles which do not containsubstantially any (e.g., about 10% or less, more typically about 5% orless, and even more typically about 1% or less) of their typical nucleicacid content.

[0068] Quantification of defective viral vector particles, empty viralvector particles, otherwise damaged particles, or any combinationthereof can be performed with any type of viral vector particle thatexhibits different emission spectrums when such particle is empty,defective, or otherwise damaged as compared to a fully intact viralvector particle, i.e., a fully processed viral vector that is not empty,defective, or otherwise damaged. Typically, such viral vector particlesexhibit a change in one or more “damage-sensitive” emission wavelengths,such as a wavelength shift and/or an intensity shift in the radiationemitted from such particles when excited. Thus, by detecting the shiftin intensity and/or wavelength the number of defective, empty, orotherwise damaged viral vector particles, or any combination thereof,can be quantified by comparison with a suitable standard signal.

[0069] A wavelength shift occurs when a damage-sensitive wavelengthcorresponding to a fully intact (undamaged) viral vector particle isreplaced by a slightly larger or smaller wavelength when the inventivemethod is practiced with a medium containing a number of defective,empty, or otherwise damaged viral vector particles. A wavelength shiftcan include any detectable shift in wavelength. Typically, a wavelengthshift will be about 20 nm or less (e.g., about 10 nm or less) inmagnitude.

[0070] The number of defective, empty, or otherwise damaged particlesdesirably is determined by an intensity shift. An intensity shift occurswhen the emitted radiation at a damage-sensitive wavelength has adetectably higher or lower intensity when emitted from defective, empty,or otherwise damaged viral vector particle versus when emitted from afully intact viral vector particle of the same type of viral vector.Whether a wavelength shift, intensity shift, or both is observed whenthe medium contains damaged viral vector particles depends on theparticular type of viral vector particle.

[0071] In quantifying defective viral vector particles, the standardsignal can be any suitable standard signal which enables relativedetermination of the quantity of defective, empty, and/or otherwisedamaged particles. For example, the standard signal can correspond to asignal produced from a medium having a relatively known amount ofdefective, empty, otherwise damaged viral vector particles, or anycombination thereof. In that respect, a crude cell lysate of viralvector particles can be enriched as to the number of defective, empty,or otherwise damaged viral vector particles, and a purified stock ofviral vector particles which contains relatively few, if any, defective,empty, or otherwise damaged particles, can be can be used to providestandard signals for comparing emission spectrums obtained from othermediums (e.g., a crude cell lysate of viral vector particles). A linearregression between the detected intensities at a damage-sensitivewavelength for the purified stock and the damage particle-enrichedlysate allows for the quantification of the number of defective, empty,or otherwise damaged viral vector particles in mediums containing moredefective, empty, or otherwise damaged viral vector particles than thepurified stock, but less than the damage particle-enriched lysate.

[0072] Quantification of the number of defective, empty, or otherwisedamaged viral vector particles in a medium can be performed under anysuitable conditions. Preferably, such techniques are performed in amedium which is at, and which has been maintained at (e.g., stored atfor a period of at least about 1 hour, at least about 12 hours, at leastabout 24 hours, at least about 1 week, or longer), a substantiallyconstant medium pH, at a substantially constant medium temperature, andfree of particle integrity-degrading detergents, to avoid undesiredintegrity changes, conformation changes, quenching, and/orphotobleaching. For example, the inventive method can be performed witha medium including or consisting of a pharmaceutical composition,maintained under the aforementioned medium conditions, which comprises astock of a viral vector, to assess whether the pharmaceuticalcomposition is suitable for administration (e.g., by examining particledegradation under such conditions). Although particularly desirable inconnection with the quantification of the number of defective, empty, orotherwise damaged viral vector particles in a medium, these conditionsalso can be useful in connection with other aspects of the inventivemethod concerning the detection and/or characterization (e.g.,quantification) of viral vector particles in a medium even in theabsence of the quantification of the number of defective, empty, orotherwise damaged viral vector particles in a medium.

[0073] Preferably, the viral vector particle also is associated with anemission wavelength that is relatively insensitive to the number ofdefective, empty, and/or otherwise damaged viral vector particles in themedium. In other words, such a wavelength and/or intensity remainsrelatively unchanged (e.g., less than about 10%, preferably less thanabout 5%, and more preferably less than about 3% changed) by thepresence of defective, empty, or otherwise damaged viral vectorparticles in the medium. The total number of viral vector particles andthe number of defective, empty, or otherwise damaged viral vectorparticles then can be relatively quantified by evaluating the emissionintensity at the insensitive wavelength and comparing it to a standardsignal, doing the same with regard to the emission intensity at thedamage-sensitive wavelength or wavelengths, and comparing the twoobtained values. The ratio of defective, empty, and/or otherwise damagedviral vector particles to the total number of particles thereby can bedetermined.

[0074] The inventive method described herein can be used to evaluate aprotocol for the production of a stock of viral vector particles, suchas a stock of a viral gene transfer vector. In such respect, a stock ofa viral vector, preferably a viral gene transfer vector, is produced inaccordance with a production protocol. The inventive method then isperformed on a medium containing the stock, or a portion thereof. Theproduction protocol is evaluated by quantifying the number of viralvector particles in the medium, the number of defective, empty, orotherwise damaged viral vector particles in the medium, or anycombination thereof.

[0075] Production protocols can be evaluated for any suitable qualityand in any suitable manner. For example, different viral vector stockproduction protocols can be compared for the total number of viralvector particles produced and/or the number of defective, empty, orotherwise damaged particles produced. Using such techniques, one candetermine the optimum factors for producing a stock, such as whatharvest time is associated with a desired particle yield of total viralvector particles (based on quantity and/or quality of the viral vectorparticles produced). Another example of a quality which can be evaluatedis the consistency of the production protocol.

[0076] The inventive method also can be used for evaluating apharmaceutical composition including a stock of a viral vector, such asa stock of a viral gene transfer vector. The pharmaceutical compositioncan be any composition containing a stock of a viral vector and asuitable pharmaceutical (e.g., physiological) carrier (such as water,with or without other additives (e.g., sugars, salts, and buffers)). Thenumber of viral vector particles in the pharmaceutical composition canbe quantified to determine whether the pharmaceutical composition issuitable for administration. For example, whether the dosage is correctcan be evaluated (e.g., whether a desired dose of viral gene transfervector particles is present). Alternatively or additionally, the numberof intact viral vector particles can be determined to assess whether thenumber and/or percentage of intact viral vector particles in thepharmaceutical composition is acceptable for administration to apatient.

[0077] The fluorescence detection methods of the invention can becombined with any number of other fluorescence detection techniques. Forexample, the viral vector particle can be assessed for its mass orshape. Mass or shape of the viral vector particle can be determinedusing techniques involving point excitation and/or point collection ofemissions, combinations of reflective mirrors, or CCD cameras, which areknown in the art. Such methods can provide an additional technique fordetermination of the quality and/or number of the viral vector particlesin the medium.

[0078] The invention also provides a method of evaluating the viralvector particle content of a medium. In this respect, a medium, whichcan be any medium described herein, is provided and contacted with anexcitation radiation having an excitation wavelength such that if aviral vector particle is in the medium an intrinsically fluorogenicportion of the viral vector particle will emit radiation having anemission wavelength at about 560-590 nm. The viral vector particlecontent (e.g., adenoviral vector content) of the medium is thenevaluated by determining whether the medium emits radiation at about560-590 nm.

[0079] This method can be used to detect the presence or absence of anyviral vector particle (such as an adenoviral vector particle) whichincludes an intrinsically fluorogenic portion that produces fluorescentemissions having an emission wavelength at about 560-590 nm, moreprecisely about 570-580 nm, and even more precisely about 574 nm, whencontacted with an excitation energy. Any suitable excitation energydescribed herein can be used. Preferably, the excitation energymaximizes viral vector particle-associated fluorescent emissions atabout 574 nm. The method also can be practiced with components of suchviral vectors, such as a viral protein or substantial homolog thereof,which has an emission wavelength of about 560-590 nm.

[0080] It has been discovered that viral vector particles can bedetected by such emission wavelengths which are significantly higherthan the emission wavelengths associated with fluorogenic amino acids(e.g., tryptophan) or nucleic acid bases (e.g., uracil). Moreover,detection at such wavelengths offers greater selectivity and possiblygreater sensitivity in detection.

[0081] As described in connection with other aspects of the presentinvention, the medium can be provided by contacting a chromatographyresin with a viral vector particle-containing composition and elutingthe viral vector particle from the chromatography resin. Preferably, ifa viral vector particle is in the composition, it will elute at a known(i.e., standard) time. The portion of the composition which wouldcontain a viral vector particle, if present, is used as the medium,thereby verifying that any detected emission wavelengths at about560-590 nm originate from the viral vector particle rather than someother fluorogenic molecule.

[0082] When the medium contains a viral vector particle, the method canfurther include quantifying the number of viral vector particles, byusing the quantification techniques described herein. Thus, for example,the intensity of the emitted radiation associated with the intrinsicallyfluorogenic portion of the viral vector particle can be determined, andthe number of viral vector particles in the medium can be quantified bycomparing the intensity of the detected radiation with a standardsignal.

[0083] The identification and/or quantification of viral vectorparticles (including the number of defective, empty, or otherwisedamaged particles) by fluorescence detection can be verified. Forexample, the method can further include detecting the mass or shape ofany fluorescent molecule in the medium emitting radiation at about560-590 nm, using techniques described herein or otherwise known in theart.

[0084] The invention further provides a method of evaluating theadenoviral vector particle content of a medium through fluorescencedetection. It has been discovered that wild-type adenoviral vectorsinclude a capsid which consists essentially of intrinsically fluorogenicproteins. Thus, any adenoviral vector containing a wild-type capsidprotein, or a substantial homolog thereof, will include an intrinsicallyfluorogenic portion. In view of these fluorogenic properties, adenoviralvector particles are particularly well suited for fluorescencedetection.

[0085] In this respect, a medium, which can be any medium as describedherein, is contacted with an excitation radiation such that, if anadenoviral vector particle is in the medium, an intrinsicallyfluorogenic portion of the adenoviral vector particle will emitradiation having an emission wavelength indicative (i.e.,characteristic) of an adenoviral vector particle. The excitationradiation can have any suitable excitation wavelength. Typically,adenoviral vectors are associated with excitation wavelengths of about220-240 nm, about 270-290 nm, or both; more precisely about 230-240 nm,about 280-290 nm, or both; and even more precisely about 235 nm, about284 nm, or both.

[0086] The adenoviral vector particle content of the medium is evaluatedby determining whether radiation having an emission wavelengthindicative of the presence or absence of an adenoviral vector particleis emitted upon contacting the medium with the excitation radiation.This determination is arrived at by comparing the detected emissionwavelengths with emission wavelengths normally associated withadenoviral vectors. Any suitable wavelength or combination ofwavelengths indicative of an adenoviral vector can be used. Typically,adenoviral vector-associated emission wavelengths include wavelengths atabout 320-340 nm, about 560-590 nm, or both; more precisely about328-332 nm, about 570-580 nm, or both; and even more precisely at about330 nm, about 574 nm, or both.

[0087] As described in connection with other aspects of the presentinvention, the medium can be provided by contacting a chromatographyresin with an adenoviral vector-containing composition and eluting theadenoviral vector from chromatography resin. Preferably, if anadenoviral vector particle is in the composition, it will elute at aknown (i.e., standard) time. The portion of the composition which wouldcontain an adenoviral vector particle, if present, is used as themedium.

[0088] When the medium contains an adenoviral vector particle, themethod can further include quantifying the number of adenoviral vectorparticles, by using the quantification techniques described herein.Thus, for example, the intensity of the emitted radiation associatedwith the intrinsically fluorogenic portion of the adenoviral vectorparticle can be determined, and the number of adenoviral vectorparticles in the medium can be quantified by comparing the intensity ofthe detected radiation with a standard signal. Preferably,quantification of adenoviral vector particles is performed using anexcitation wavelength of about 270-295 nm (e.g., 284 nm) and an emissionwavelength of about 560-590 nm (e.g. 574 nm).

[0089] The identification and/or quantification of adenoviral vectorparticles (including the number of damaged particles) by fluorescencedetection can be verified. For example, the method can further includedetecting the mass or shape of any fluorescent molecule in the mediumemitting radiation at wavelengths indicative of adenoviral vectorparticles, using techniques described herein or otherwise known in theart.

[0090] The inventive methods also can include separation andidentification of the intrinsically fluorogenic portion of the viralvector particle or components thereof The fluorogenic portion and itscomponents can be separated by any suitable method. For example, thecomponents of a chemically disassociated viral vector particle can beelectrophoretically separated, based on size and/or charge. Preferably,separation of the fluorogenic portion or its components is performed byreverse phase chromatography. These separated components can besubjected to fluorescence detection to identify and/or characterize thefluorogenic portion of the viral vector particle.

[0091] The invention additionally provides a method of evaluating theintrinsically fluorogenic adenoviral structural protein content of amedium. An intrinsically fluorogenic adenoviral structural protein isany adenoviral protein that is a wild-type adenoviral protein or asubstantial homolog, which is intrinsically fluorogenic, and whichnormally forms a part of, or which can be made a part of, an adenoviralcapsid. Such proteins can be obtained, for example, by performingreverse phase chromatography, or other separation methods discussedherein or known in the art, on an adenoviral vector particle.Alternatively, such proteins can be produced by any other suitabletechnique (e.g., by recombinant DNA technology).

[0092] In this respect, a medium is provided, which can be any mediumdiscussed herein, and contacted with an excitation radiation having anexcitation wavelength, such that if an intrinsically fluorogenicadenoviral structural protein is in the medium it will emit radiationhaving an emission wavelength characteristic of an intrinsicallyfluorogenic adenoviral structural protein. The adenoviral proteincontent of the medium is evaluated by determining whether radiationhaving an emission wavelength characteristic of an intrinsicallyfluorogenic adenoviral structural protein is emitted from the medium.

[0093] The medium can be provided by the use of chromatography toseparate a composition containing an intrinsically fluorogenicadenoviral structural protein, such that an eluted portion of thecomposition will contain an adenoviral vector structural protein, ifpresent, as described herein. Moreover, the method can includequantification of the number of adenoviral structural proteins in themedium. The intrinsically fluorogenic adenoviral structural protein canbe part of a larger complex, for example, a complex of proteins, or evenpart of a different type of vector.

[0094] Numerous alternative and equivalent techniques and devices tothose described herein as useful in the context of the present inventionare possible. Several of such techniques and devices are known in theart, and are described in, for example, Brand, L. and Johnson, M. L.,Eds., Fluorescence Spectroscopy (Methods in Enzymology, Volume 278),Academic Press (1997); Cantor and Schimmel, Biophysical Chemistry, W. H.Freeman & Co. (New York) (11th Printing 1998), Dewey, T. G., Ed.,Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, PlenumPublishing (1991); Guilbault, G. G., Ed., Practical Fluorescence, SecondEdition, Marcel Dekker (1990); Lakowicz, J. R., Ed., Topics inFluorescence Spectroscopy: Techniques, Volumes 1-5 (1991); PlenumPublishing; Lakowicz, J. R., Principles ofFluorescence Spectroscopy,Second Edition, Plenum Publishing (1999); and Sharma, A. and Schulman,S. G., Introduction to Fluorescence Spectroscopy, John Wiley and Sons(1999).

EXAMPLES

[0095] The following examples further illustrate the present inventionbut should not be construed as in any way limiting its scope.

Example 1

[0096] This example demonstrates the identification of excitation andemission wavelengths for the intrinsically fluorogenic portion of aviral vector particle.

[0097] A 100 μl solution containing approximately 1.3×10⁹ anion exchangechromatography-purified, wild-type adenovirus particles (serotype 5) wasobtained. No fluorescent dyes or fluorophores were in, or added to, thesolution. The solution was placed in a Hewlett Packard 1100 scanningFluorescence Detector, equipped with a xenon flash excitation radiationsource and a PMT detector. The solution was scanned to determine whetherthe adenovirus particles were intrinsically fluorogenic by contactingthe solution with UV radiation emitted from the xenon flash lamp atwavelengths between 220 nm and 250 nm. An emission peak was detectedwhen the solution was contacted with excitation radiation having anexcitation wavelength at about 235 nm. Thus, it was determined that theadenovirus particles contain a naturally-occurring intrinsicallyfluorogenic portion.

[0098] Emission wavelengths then were scanned by fixing the excitationradiation at 235 nm (+/−10 nm), contacting the solution with theexcitation radiation, and detecting whether fluorescent emissions havingemission wavelengths of between 300 nm and 600 nm were produced. Aprominent emission wavelength was observed at about 330 nm.

[0099] Excitation wavelengths were re-scanned by fixing detectedemission wavelengths at 330 nm and scanning excitation wavelengths from200 nm to 300 nm. Two excitation wavelengths, one at about 234 nm andanother at about 284 nm, which resulted in significant fluorescentemissions at 330 nm, were observed.

[0100] Emission wavelengths were then re-scanned by fixing theexcitation wavelength at 281 nm (+/−10 nm) and detecting whetherfluorescent emissions having emission wavelengths of between 300 nm and700 nm were produced. Two emission wavelengths were observed: one atabout 330 nm, and, surprisingly, a second emission wavelength at about574 nm, which is well above emission wavelengths associated witharomatic amino acids (282-348 nm), pyrimidine nucleotide bases (260-275nm), and purine nucleotide bases (260-267 nm). Fluorescent emissions atthe 574 nm emission wavelength were determined to be more selective forthe adenovirus particles (i.e., emissions at this wavelength tended tobe associated with less background fluorescence and/or undesiredexcitation of other molecules in the solution) and resulted insignificantly higher emission intensity than at 330 nm, indicating thatthis emission wavelength was the optimum wavelength, and, thus, alsocapable of providing the most sensitive detection.

[0101] The results of these experiments demonstrate how excitationwavelengths and emission wavelengths can be determined for a viralvector particle containing an intrinsically fluorogenic portion, such asan adenoviral vector particle. The results also demonstrate that viralvector particles which are associated with more than one excitationemission wavelength can be detected using the method of the invention,and that such wavelengths can be exploited to provide more sensitiveand/or more selective viral vector particle detection. Furthermore,these results demonstrate that viral vector particles having an emissionwavelength of between 560-590 nm can be detected by fluorescentdetection.

Example 2

[0102] This example demonstrates the relationship between emissionintensity and total number of adenoviral vector particles in a medium.

[0103] A medium containing 3.84×10⁹ unmodified serotype 5 adenovirusparticles was subjected to six repeated 1:3 serial dilutions. At eachdilution of the medium, three 100 μl samples of the diluted medium wereobtained and subjected to excitation radiation at 235 nm andfluorescence detection using a Hewlett Packard 1100 FluorescenceDetector, as described in Example 1. The excitation radiation wavelengthwas set at 235 nm.

[0104] Emission radiation having an emission wavelength of 330 nm andthe corresponding intensity of the emission wavelength for each of thesamples was detected by a PMT contained in the fluorescence detector.Intensity was measured in relative light units (RLUs). An emissionspectrum corresponding to detected emission wavelengths and intensitieswas produced. The area under each peak in the emission spectrum wasintegrated by ChemStation 3D version 8.0 (Hewlett Packard). Integratedpeak areas for each sample at each dilution were averaged except forresults which varied more than 5% from the mean peal area at a givendilution which were not considered. The results of these experiments arepresented in Table 2. TABLE 2 Adenovirus Particle Number andFluorescence Intensity Estimated Number of Adenovirus Particles in theDiluted Medium Fluorescence Intensity (RLU) 5.27 × 10⁶ 9.6 1.58 × 10⁷ 264.74 × 10⁷ 84 1.42 × 10⁸ 273 4.27 × 10⁸ 876 1.28 × 10⁹ 2525

[0105] These results were plotted on a graph of the number of adenovirusparticles in the diluted medium versus the intensity of the fluorescenceintensity. The plotted data formed a line. The linear nature of therelationship between viral vector particle number and fluorescentintensity was confirmed by linear regression analysis. The regressioncoefficient was determined to be 0.9998.

[0106] These results show that mediums containing viral vector particleswhich include an intrinsically fluorogenic portion exhibit fluorescenceintensity in a linear relationship to the number of viral vectorparticles in the medium, and, thus, are subject to quantification byfluorescent detection. The results further demonstrate that this linearrelationship extends across a wide range of total viral vector particlenumbers (e.g., from about 5.27×10⁶ particles to about 1.28×10⁹particles). Thus, the present invention provides a method of quantifyingthe number of viral vector particles in a medium by fluorescentdetection.

Example 3

[0107] This example demonstrates numerous aspects of the presentinventive method including the increased sensitivity of the inventivemethod over presently used UV spectrophotometry-based techniques, aswell as the relative quantification of intact adenovirus particles andthe relative quantification of defective and empty adenovirus particlesby fluorescence detection in various mediums.

[0108] Cells infected with unmodified serotype 5 adenovirus wereharvested to obtain a crude cell lysate using standard techniques. Asample of the crude cell lysate was obtained.

[0109] Two additional samples were prepared as follows: Two aliquots ofthe remaining cell lysate were obtained and subjected to purification bycontact with an anion exchange high performance liquid chromatographycolumn (AE-HPLC) as described in International Patent Application WO99/54441, or by separation on a cesium chloride density gradientrepeated three times (i.e., triple, or 3×, CsCl density gradientpurification). Volumes equal to the crude lysate sample were obtainedfor both purified samples.

[0110] Yet two more samples were prepared as follows: Another aliquot ofthe cell lysate was subjected to AE-HPLC purification followed by onetime (i.e., 1×) CsCl density gradient purification. After 1×CsCl densitygradient purification, two distinct bands on the AE-HPLC column eluant(an upper and lower band), corresponding to the purified AE-HPLC eluantwere observable. Samples extracted from each band, in a volume equal tothe aforementioned samples, were obtained to provide an upper bandsample and a lower band sample. Mass spectrometry analysis determinedthat the upper band sample contained higher quantities of incompletelyprocessed (i.e., empty and defective) adenovirus particles than thelower band sample, and even more defective and empty particles than thecrude cell lysate.

[0111] Each of the aforementioned five samples was divided into threeequal volumes. The equal volumes were separately injected into ananalytical chromatography column which was connected to both a UVspectrophotometer and the fluorescence detector described in Example 1.Thus, the portions of the samples eluted from the analytical column weresubjected to UV spectrophotometry-based absorbance analysis as well asexcitation and fluorescence detection almost immediately followingelution. The time of elution from the analytical column and absorbanceof fluorescence detection were determined. Elution times for virusesdetected by absorbance and excitation varied by 1 second or less.

[0112] Absorbance quantification was performed at 260 nm for eachportion eluted from the analytical column using standard techniques. Theeluant obtained from the first equal volume of each sample was contactedwith excitation radiation having an excitation wavelength (Ex) of 235nm. This was followed by fluorescence detection at the 330 nm emissionwavelength (Em). The eluants obtained from the second and third equalvolumes of each sample were contacted with excitation radiation at 284nm, which was followed by fluorescence detection at either the 330 nm or574 nm emission wavelength, respectively. Fluorescent intensity in allcases was measured in relative light units.

[0113] Absorbance and fluorescence intensities for adenovirus particlesin each eluted portion were separately plotted against time of elutionto provide two dimensional graphs containing absorbance or emissionpeaks. Integrated peak areas for absorbance, and for fluorescenceintensity corresponding to each of the excitation and emissionwavelength combinations used (i.e., 235 nm Ex: 330 nm Em, 284 nm Ex: 330nm Em, and 284 nm Ex: 574 nm Em), were determined. Normalized values foreach portion were determined by dividing the integrated area of thefluorescence peaks by the integrated area of the absorbance peaks. Theresults of these calculations are shown in Table 3. TABLE 3 NormalizedFluorescence/Absorbance Values for Adenoviral Vector Particles inVarious Meduims Fluorescence Fluorescence Fluorescence Intensity PeakIntensity Peak Intensity Peak Area (Ex = 235 Area (Ex = 284 Area (Ex =284 nm: Em = 330 nm: Em = 330 nm: Em = 574 nm)/Absorbance nm)/Absorbancenm)/Absorbance Peak Area (260 Peak Area (260 Peak Area (260 nm) nm) nm)Crude cell lysate 16.0 22.3 14.4 AE-HPLC purified 16.0 22.3 20.5 lysate3x CsCl density- 14.3 20.2 19.6 gradient purified lysate AE-HPLCpurified + 14.2 19.5 19.1 1 × CsCl density gradient purification (lowerband) AE-HPLC purified + 15.0 21.1 13.1 1 × CsCl density gradientpurification (upper band)

[0114] The results of these experiments are significant in manyrespects. First, the normalized values in Table 3 are indicative of therelative sensitivity of the inventive method as compared to UVspectrophotometry-based absorbance analysis.

[0115] Particularly, as can be seen from the data set forth in Table 3,the fluorescence detection methods of the present invention are moresensitive than detection methods based on UV absorbance.

[0116] Second, the results of these experiments demonstratequantification of the number of adenovirus particles in a medium by theinventive method. For example, the lower band sample, which containedonly a portion of the eluant obtained from the AE-HPLC purificationstep, exhibited significantly lower normalized values than crude celllysate and AE-HPLC purified samples at the 330 nm emission wavelength.These lower normalized values reflect lower fluorescence intensity atthe 330 nm emission wavelength emitting from the lower band sample, and,consequently, reflect a smaller number of viral particles in the lowerband sample versus the crude cell lysate and AE-HPLC purified samples,as predicted. Thus, these results confirm that the present inventionprovides a method for relatively quantifying the number of viral vectorparticles in a medium by fluorescence detection. The consistentnormalized values obtained at 330 nm emission wavelengths for theAE-HPLC purified sample and crude cell lysate sample indicate thatnearly all of the particles were retained by the AE-HPLC purificationtechnique and corroborate the ability of the inventive method toquantify the number of viral particles in the medium.

[0117] Third, these results demonstrate the relative quantification ofdefective and empty viral vector particles in a medium by fluorescencedetection. For example, as seen in Table 3, the crude cell lysate sampleexhibited a significantly lower normalized value, and, thus, lowerfluorescence intensity, at the 574 nm emission wavelength, than theAE-HPLC purified sample. The upper band sample exhibited an even moresignificant decrease in normalized value at the 574 nm emissionwavelength as compared to the AE-HPLC purified sample. The decrease influorescence intensity at the 574 nm emission wavelength observed in thecrude cell lysate and upper band samples versus the AE-HPLC purifiedsample corresponds to the higher number of defective and empty particlesin these samples, as confirmed by mass spectrometry experiments.Similarly, the relatively higher fluorescence intensity at the 547 nmemission wavelength observed for the lower band sample versus theAE-HPLC purified sample corresponds to the lower number of defective andempty particles in this sample, as also confirmed by mass spectrometryexperiments.

[0118] Using fluorescent emissions at the 574 nm emission wavelengthfrom the upper band as a standard signal, and by performing a linearregression analysis, the relative percentage of empty and defectiveparticles in other samples was determined. For example, by performingsuch an analysis it was determined that the AE-HPLC sample containedabout 11.5% of the number of empty and defective particles contained inthe upper band sample.

[0119] The results of these experiments also confirm that the 330 nmemission wavelength is relative damage-insensitive for adenovirusparticles and that the 574 nm emission wavelength is a damage-sensitiveemission wavelength for adenovirus particles, which is associated withan intensity shift. The relative consistency of fluorescence intensityat the 330 nm emission wavelength for the various samples confirms thatthis wavelength is a relatively damage-insensitive wavelength. Therelative variance of fluorescence intensity at the 574 nm emissionwavelength between the various samples known to differ as to thequantity of damaged viral vector particles confirms that this wavelengthis a damage-sensitive wavelength for adenovirus particles. Thus, thenormalized value (or fluorescence intensity) observed at the 330 nmemission wavelength can be used to calculate the total number ofparticles, and the value obtained at the 574 nm emission wavelength candetermine what proportion of those particles are damaged.

[0120] This experiment demonstrates that the present inventive methodcan be used to quantify the number of viral vector particles in a mediumand/or quantify the number of damaged (e.g., defective and empty) viralvector particles in a medium.

Example 4

[0121] This example demonstrates the separation and identification ofthe intrinsically fluorogenic components of adenovirus particles and theevaluation of the intrinsically fluorogenic adenoviral structuralprotein content of a medium.

[0122] A first solution containing 1.9×10⁹ intact wild-type serotype 5adenovirus particles and a second solution containing 3.5×10¹⁰ intactwild-type serotype 5 adenovirus particles were obtained using standardtechniques. Each solution was subjected to C4 reverse phasechromatography without disassociation of the adenovirus particles priorto contacting the reverse phase column with the solutions. Theadenovirus particles were separated into their constituent molecules bycontact with the reverse phase chromatography resin, and the separatedcompounds were eluted from the resin. The time of elution of thecomponents was determined.

[0123] After elution, the eluted portion of the second solution wassubjected to UV spectrophotometry at 214 nm to identify the separatedcomponents of the adenovirus particles. The resulting absorbance wasplotted on a graph against the time of elution of the components. Peakarea was determined to identify the proportion of the total adenovirusprotein content corresponding to each detected component.

[0124] Similarly, after elution, the eluted portion of the firstsolution was subjected to excitation radiation having an excitationwavelength at 235 nm and fluorescent detection for emissions having anemission wavelength at 330 nm using the techniques described in Example1 to determine which of the identified components were intrinsicallyfluorescent. Fluorescence intensity was plotted on a graph against thetime of elution of the fluorescent components. Peak area was determinedto identify the proportion of the fluorescence intensity of eachfluorogenic component to the combined fluorescence for all components,thereby enabling determination of the relative fluorescence of theintrinsically fluorogenic components.

[0125] Significant absorbance peaks were observed for components elutedfrom the resin at about 9.5 minutes, 10 minutes, 10.5 minutes, 11.5minutes, 12 minutes, 12.5 minutes, 14 minutes, 15 minutes, 22 minutes,and 24 minutes. These peaks were correlated with fluorescence peaksobserved for components eluted from the resin at about 2.5 minutes, 12minutes, 13.5 minutes, 15.5 minutes, 16 minutes, 18 minutes, 19 minutes,26 minutes, and 28 minutes. Other peaks were determined to be caused bydefective or empty adenovirus particles or buffers in the solution andwere not further analyzed. Correlation of the detected components wasconfirmed, and molecules identified, by enzymatic digestion and massspectrometry analysis (including amino acid sequencing) of the elutedcomponents for both solutions.

[0126] By comparing the resulting absorbance and fluorescence spectrums,it was determined that the absorbance peak corresponding to thecomponent eluted at 9.5 minutes was not represented by a correspondingpeak in the fluorescence spectrum. By mass spectrometry analysis it wasdetermined that this component consists of the N-terminal portion of theadenovirus major core protein (protein VII). In contrast, each of theother detected components were determined to fluoresce when contactedwith excitation radiation at 235 nm.

[0127] Comparisons were made between the areas of the absorbance peaksand the fluorescence peaks to determine the relative component contentof the elution and its proportional contribution to the totalfluorescence of the solution. By making such comparisons the fluorescentqualities of the components was relatively determined. For example, theadenovirus protein hexon, which eluted at 22 minutes in solution, wasdetermined by its absorbance peak area to make up 52% of the totalprotein content of the adenovirus particles (consistent with publishedfigures), whereas its fluorescent emission contribution was about 75% ofthe total detected fluorescence of the components. Thus, hexon wasidentified as a strong intrinsically fluorogenic adenoviral protein.Other intrinsically fluorogenic adenovirus structural proteinsidentified by these experiments include the adenovirus fiber and pentonproteins. Similar emission patterns were seen in other experimentsperformed using excitation radiation having an excitation wavelength of284 nm and fluorescence detection at 330 nm.

[0128] These results demonstrate that components of intrinsicallyfluorogenic portions of viral vector particles can be separated andevaluated by fluorescence detection. Moreover, these results demonstratethat the adenoviral vector structural protein content of a medium can bedetermined by fluorescence detection.

[0129] All references, including publications, patent applications andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein. The use of the terms “a” and “an” and “the” and similarreferents in the context of describing the present invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the terms “including,”“having,” “comprising,” “containing, ” and similar terms are to beconstrued as open-ended terms (i.e., meaning “including, but not limitedto”) unless otherwise indicated. Recitation of ranges of values hereinare merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. The use ofany and all examples, or exemplary language (e.g., “such as”) providedherein, is intended merely to better illustrate the present inventionand is not intended as a limitation on the scope of the claimedinvention. No language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention.

[0130] The foregoing is an integrated description of the invention as awhole, not merely of any particular element or facet thereof. Thedescription describes “preferred embodiments” of this invention,including the best mode known to the inventors for carrying it out. Uponreading the foregoing description, variations of those preferredembodiments may become apparent to those of ordinary skill in the art.The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is possible unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of quantifying the number of viralvector particles in a medium, the method comprising: (a) providing amedium containing at least one viral vector particle comprising anintrinsically fluorogenic portion, (b) contacting the medium with anexcitation energy such that an electron associated with theintrinsically fluorogenic portion is raised to an excited energy state,(c) permitting the excited electron to emit radiation having an emissionwavelength and an intensity, (d) detecting the emission wavelength andthe intensity, (e) providing a standard signal, and (f) quantifying thenumber of viral vector particles in the medium by comparing the emissionwavelength and the intensity detected in step (d) with the standardsignal.
 2. The method of claim 1, wherein the method further comprisesquantifying the number of defective viral vector particles in themedium, the number of empty viral vector particles in the medium, orboth.
 3. The method of claim 1, wherein the medium is provided bycontacting a chromatography resin with a composition comprising a viralvector particle and eluting a portion of the composition containing theviral vector particle from the chromatography resin such that the elutedportion is the medium.
 4. The method of claim 1, wherein the mediumcontains about 1×10⁹ viral vector particles/ml or less.
 5. The method ofclaim 4, wherein the medium contains about 5×10⁷ viral vectorparticles/ml or less.
 6. The method of claim 1, wherein the methodcomprises exciting electrons associated with the intrinsicallyfluorogenic portion while substantially not exciting electronsassociated with any other fluorogenic molecules in the medium.
 7. Themethod of claim 1, wherein the viral vector is an adenoviral vector. 8.The method of claim 1, wherein the medium is a pharmaceuticalcomposition comprising a stock of a viral gene transfer vector, and themethod further comprises evaluating whether the pharmaceuticalcomposition is suitable for administration.
 9. The method of claim 8,wherein the method further comprises quantifying the number ofnon-intact viral gene transfer vector particles in the pharmaceuticalcomposition, wherein the pharmaceutical composition has been maintainedat a substantially constant pH, at a substantially constant temperature,and free of integrity-degrading detergents for a period of at leastabout 1 hour prior to, and during, the method.
 10. The method of claim1, wherein the method further comprises quantifying the number ofdefective viral vector particles in the medium, the number of emptyviral vector particles in the medium, or both.
 11. A method ofevaluating a protocol for the production of a viral vector stockcomprising: (i) producing a stock of a viral vector in a medium inaccordance with a production protocol, (ii) performing the method ofclaim 1 on the medium containing the stock, or a portion thereof, and(iii) evaluating the protocol by quantifying the number of viral vectorparticles in the medium or portion thereof.
 12. The method of claim 11,wherein the viral vector is a viral gene transfer vector.
 13. The methodof claim 11, wherein the method further comprises quantifying the numberof defective viral vector particles in the medium or portion thereof,quantifying the number of empty viral vector particles in the medium orportion thereof, or both.
 14. A method of evaluating the viral vectorparticle content of a medium, the method comprising: (a) providing ,amedium, (b) contacting the medium with an excitation radiation having anexcitation wavelength such that if a viral vector particle is in themedium an intrinsically fluorogenic portion of the viral vector particlewill emit radiation having an emission wavelength at about 560-590 nm,and (c) evaluating the viral vector particle content of the medium bydetermining whether the medium emits radiation at about 560-590 nm. 15.The method of claim 14, wherein providing the medium comprises: (i)contacting a composition with a chromatography resin, (ii) eluting aportion of the composition from the chromatography resin such that if aviral vector particle is in the composition it will elute from thechromatography resin at a known time, and (iii) obtaining the portion ofthe composition which will contain a viral vector particle, if presentin the composition, to provide the medium.
 16. The method of claim 14,wherein the medium contains at least one viral vector particle having anintrinsically fluorogenic portion, and wherein the method furthercomprises: (d) determining the intensity of the emitted radiation atabout 560-590 nm, (e) providing a standard signal, and (f) quantifyingthe number of viral vector particles in the medium by comparing theintensity of the emitted radiation determined in step (d) with thestandard signal.
 17. The method of claim 16, wherein step (d) of themethod comprises determining the intensity of the emitted radiation atabout 574 nm.
 18. A method of evaluating the adenoviral vector particlecontent of a medium, the method comprising: (a) providing a medium, (b)contacting the medium with an excitation radiation having an excitationwavelength such that if an adenoviral vector particle is in the mediuman intrinsically fluorogenic portion of the adenoviral vector particlewill emit radiation having an emission wavelength characteristic of awild-type adenoviral vector particle or a substantial homolog thereof,and (c) evaluating the adenoviral vector particle content of the mediumby determining whether the medium emits radiation characteristic of awild-type adenoviral vector particle or a substantial homolog thereof.19. The method of claim 18, wherein the excitation radiation has anexcitation wavelength of about 235 nm, about 284 nm, or both, and step(c) comprises determining whether the medium emits radiation having anemission wavelength of about 330 nm, about 574 nm, or both.
 20. Themethod of claim 18, wherein providing the medium comprises: (i)contacting a composition with a chromatography resin, (ii) eluting aportion of the composition from the chromatography resin such that if anadenoviral vector particle is in the composition it will elute from thechromatography resin at a known time, and (iii) obtaining the portion ofthe composition which will contain an adenoviral vector particle, ifpresent in the composition, to provide the medium.
 21. The method ofclaim 18, wherein the medium contains at least one adenoviral vectorparticle having an intrinsically fluorogenic portion, and wherein themethod further comprises: (d) determining the intensity of the emittedradiation associated with the intrinsically fluorogenic portion of theadenoviral vector particle, (e) providing a standard signal, and (f)quantifying the number of adenoviral vector particles in the medium bycomparing the intensity of the emitted radiation determined in step (d)with the standard signal.
 22. A method of evaluating the intrinsicallyfluorogenic adenoviral structural protein content of a medium, themethod comprising: (a) providing a medium, (b) contacting the mediumwith an excitation radiation having an excitation wavelength, such thatif an intrinsically fluorogenic adenoviral structural protein is in themedium it will emit radiation having an emission wavelengthcharacteristic of an intrinsically fluorogenic adenoviral structuralprotein, and (c) evaluating the intrinsically fluorogenic adenoviralstructural protein content of the medium by determining whether themedium emits radiation having an emission wavelength characteristic ofan intrinsically fluorogenic adenoviral structural protein.
 23. Themethod of claim 22, wherein providing the medium comprises: (i)contacting a composition with a chromatography resin, (ii) eluting atleast a portion of the composition from the chromatography resin suchthat if an intrinsically fluorogenic adenoviral structural protein is inthe composition it will elute at a known time, and (iii) obtaining theportion of the composition which will contain an intrinsicallyfluorogenic adenoviral structural protein, if present in thecomposition, to provide the medium.
 24. The method of claim 22, whereinthe medium contains at least one intrinsically fluorogenic adenoviralstructural protein, and wherein the method further comprises: (e)determining the intensity of the emitted radiation associated with theintrinsically fluorogenic adenoviral structural protein in the medium,(d) providing a standard signal, and (f) quantifying the number ofintrinsically fluorogenic adenoviral structural proteins in the mediumby comparing the intensity of the emitted radiation detected in step (d)with the standard signal.